Novel technetium and rhenium complexes

ABSTRACT

Novel radioactive technetium and rhenium complexes comprising tripodal ligands are provided by the present invention. In particular, Tc(I) complexes are provided by the present invention. Novel ligands suitable for the formation of the technetium and rhenium complexes of the invention are also provided, as well as radiopharmaceutical compositions comprising said complexes, kits for their preparation. The invention also relates to the use of  99m Tc radiopharmaceuticals of the invention for SPECT imaging.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/576,543, filed Apr. 3, 2007, now U.S. Pat. No. 8,022,211, which is afiling under 35 U.S.C. 371 of international application numberPCT/GB2005/003806, filed Oct. 4, 2005, which claims priority toapplication number 0421987.9 filed Oct. 4, 2004, in Great Britain, theentire disclosure of which is hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to radioactive technetium (Tc) and rhenium(Re) complexes and in particular Tc complexes based on technetium(I)[Tc(I)]. Novel Tc and Re complexes are provided by the present inventionas well as a number of novel ligands suitable for use in forming saidcomplexes. The present invention additionally relates toradiopharmaceutical products comprising the novel Tc and Re complexesand kits for the facile preparation of said radiopharmaceuticals.Various uses of these radiopharmaceutical products are also covered bythe present invention.

DESCRIPTION OF RELATED ART

The coordination chemistry of Tc and Re plays an important role in thedesign and synthesis of new radiopharmaceuticals for nuclear medicine.^(99m)Tc radiopharmaceuticals have widespread application in the fieldof nuclear medicine. The physical properties of ^(99m)Tc make it one ofthe most desirable radionuclides for external imaging. It is readilyavailable from ⁹⁹Mo—^(99m)Tc generator systems, has a half life of 6hours and an optimal energy gamma emission of 140 keV. Re complexes arestructural models of the Tc complexes, since the homologous compoundsdisplay very similar coordination parameters and physical properties. Inaddition, Re complexes with the nuclei ¹⁸⁶Re and ¹⁸⁸Re have attractiveproperties because they are β-emitters with therapeutically-usefulenergy. A wide range of complexes with these metals have been reported,some of which are currently used in nuclear medicine.

Radiopharmaceuticals based on Tc(I) are desirable due to the fact thatthe d⁶-low spin Tc(I) ion is kinetically inert, meaning that suchradiopharmaceuticals are extremely stable in vivo. For many years, theonly important radiopharmaceutical with Tc in a low oxidation state was^(99m)Tc[MIBI]₆ ⁺, which is stable in vivo, but not very suitable forchemical substitution. More recently, the development of additionalTc(I) radiopharmaceuticals has been supported by the elucidation of asimple and rapid procedure for the preparation of a reactive Tc(I)moiety, thus facilitating extensive organometallic chemistry. Thisreactive Tc(I) moiety is [Tc(CO)₃(H₂O)₃]⁺ and was reported by Alberto etal [J. Am. Chem. Soc. 120 pp 7987-8 (1998)]. The three water moleculesbound to the highly inert fac-[Tc(CO₃)]⁺ core can be readily substitutedby a number of functional groups to cap one face of the Tc(I) ion andproduce stable complexes containing the Tc(CO)₃ moiety. For example,Kothari et al [Appl. Rad. Isot. 58 p543-9 (2003)] demonstrated that anNP₂ ligand reacts with [Tc(CO)₃(H₂O)₃]⁺ to form a stable chelate.

It would be advantageous if the further development of technetium andrhenium radiopharmaceuticals led to the provision of stable metalcomplexes that are (i) readily coupled to a range of biological vectors,(ii) easily modified to produce both neutral and unipositively chargedmetal complexes, and (iii) readily derivatised in order to modify boththe electronic properties and solubility of the metal complexes.

The tripodal ligand tris(2-pyridyl)methylamine, and some of itsderivatives, have been reported by Arnold et al [JCS Dalton Trans. pp736-46 (2001)]. The paper discloses complexes of the ligands withcopper(II) and zinc(II) along with their crystal structures todemonstrate the coordination modes of the various complexes. There is nosuggestion by Arnold that the ligands disclosed would be useful in theprovision of radioactive technetium and rhenium complexes.

SUMMARY OF THE INVENTION

Novel technetium and rhenium complexes comprising tripodal ligands areprovided by the present invention. The tripodal ligands used in thepresent invention form metal complexes displaying superior propertiesover prior art metal complexes.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a metal complex of theligand of Formula I:

wherein:

the metal is a radioactive isotope of either technetium or rhenium;

R¹ is an E group or —CH₂(AR⁵), wherein A is S or O and R⁵ is H or C₁₋₆alkyl;

R² and R³ are each independently an E group;

E is an optionally substituted C₃₋₁₀ nitrogen-containing heteroarylring; and,

R⁴ is —(Z)_(q)—Y;

where each Z is independently —CQ₂-, —CQ=CQ-, —C≡C—, —CQ₂CO₂—, —CO₂CQ₂-,—NQ-, —NQCO—, —CONQ-, —NQ(C═O)NQ-, —NQ(C═S)NQ-, —SO₂NQ-, —NQSO₂—,—CQ₂OCQ₂-, —CQ₂SCQ₂-, —CQ₂(NQ)CQ₂-, a C₄₋₈ cycloheteroalkylene group, aC₄₋₈ cycloalkylene group, a C₅₋₁₂ arylene group, or a C₃₋₁₂heteroarylene group, an amino acid or a monodisperse polyethyleneglycol(PEG) building block; wherein each Q is independently selected from H,C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxyalkyl or C₁₋₄hydroxyalkyl;

q is an integer of value 0 to 10; and

Y is H, amino, C₁₋₁₀ alkyl, C₃₋₂₀ alkylaryl, C₆₋₁₄ aryl, C₃₋₁₂heterocycle, C₂₋₁₀ alkoxyalkyl, C₁₋₁₀ hydroxyalkyl, C₁₋₁₀ aminoalkyl,C₁₋₁₀ hydroxyalkyl, C₁₋₁₀ alkoxy, C₁₋₁₀ acyloxy, C₆₋₁₀ aryloxy, or C₆₋₁₀aroyloxy, or Y is a biological targeting moiety; and wherein not morethan three out of a possible five atoms of the ligand are involved incomplexation.

A “metal complex” as defined in the present invention means a relativelystable coordination complex wherein the ligand is of Formula I and thecentral metal is an isotope of Tc or Re. When the metal is an isotope ofTc, it is preferably ^(99m)Tc or ^(94m)Tc, in which case the Tc complexof the invention is suitable for in vivo diagnostic imaging by singlephoton emission computed tomography (SPECT) or positron emissiontomography (PET), respectively. When the metal is an isotope of Re, itis preferably ¹⁸⁶Re or ¹⁸⁸Re and therefore suitable for therapy. It isstrongly preferred that the metal complex is “resistant totranschelation”, i.e. does not readily undergo ligand exchange withother potentially competing ligands for the metal coordination sites.

A “nitrogen-containing heteroaryl ring” of the present invention is anymonocyclic or fused aromatic ring structure comprising at least onenitrogen atom, preferably one or two N atoms, most preferably a single Natom. Preferable nitrogen heteroaryl groups of the present inventioncomprise 5- and 6-membered nitrogen-containing ring structures, examplesof which are pyridyl, imidazoyl, pyrimidine, pyrrole, oxazole andpyrazoyl rings plus fused ring analogues thereof such as quinoline. Theterm “substituted” in the context of nitrogen-containing heteroarylrings means that one or more organic substituents is attached to thenitrogen-containing heteroaryl ring either at the nitrogen or elsewherein the ring. Examples of suitable organic substituents include C₁₋₆alkyl, amino, nitro, halo, carboxy, sulphonate and C₆₋₁₄ aryl.

A “biological targeting moiety” is defined in the present invention as amolecule which functions as a means to transport the metal complex to aparticular site within the mammalian body, when said metal complex is aradiopharmaceutical. Preferably, the site within the mammalian body isthe site of a particular disease state. Suitable biological targetingmoieties include: 3-100 mer peptides or peptide analogues which may belinear peptides or cyclic peptides or combinations thereof; monoclonalantibodies or fragments thereof; or enzyme substrates or inhibitors;synthetic receptor-binding compounds; oligonucleotides, or oligo-DNA oroligo-RNA fragments. The biological targeting moiety may be of syntheticor natural origin, but is preferably synthetic. By the term “synthetic”is meant the conventional meaning of the term i.e. man-made as opposedto being isolated from natural sources e.g. from the mammalian body.Such compounds have the advantage that their manufacture and impurityprofile can be fully controlled. By the term “cyclic peptide” is meant asequence of 5 to 15 amino acids in which the two terminal amino acidsare bonded together by a covalent bond which may be a peptide ordisulphide bond or a synthetic non-peptide bond such as a thioether,phosphodiester, disiloxane or urethane bond.

By the term “amino acid” is meant an L- or D-amino acid, amino acidanalogue or amino acid mimetic which may be naturally occurring or ofpurely synthetic origin, and may be optically pure, i.e. a singleenantiomer and hence chiral, or a mixture of enantiomers. Preferably theamino acids of the present invention are optically pure. By the term“amino acid mimetic” is meant synthetic analogues of naturally occurringamino acids which are isosteres, i.e. have been designed to mimic thesteric and electronic structure of the natural compound. Such isosteresare well known to those skilled in the art and include but are notlimited to depsipeptides, retro-inverso peptides, thioamides,cycloalkanes or 1,5-disubstituted tetrazoles [see M. Goodman,Biopolymers, 24, 137, (1985)].

Suitable peptides for use in the present invention include:

-   -   somatostatin, octreotide and analogues,    -   peptides which bind to the ST receptor, where ST refers to the        heat-stable toxin produced by E. coli and other micro-organisms;    -   laminin fragments e.g. YIGSR (SEQ ID NO:1), PDSGR (SEQ ID NO:2),        IKVAV (SEQ ID NO:3), LRE and KCQAGTFALRGDPQG (SEQ ID NO:4),    -   N-formyl peptides for targeting sites of leucocyte accumulation,    -   Platelet factor 4 (PF4) and fragments thereof,    -   RGD (Arg-Gly-Asp)-containing peptides, which may e.g. target        angiogenesis [R. Pasqualini et al., Nat. Biotechnol. 1997 June;        15(6):542-6]; [E. Ruoslahti, Kidney Int. 1997 May;        51(5):1413-7].    -   peptide fragments of α₂-antiplasmin, fibronectin or beta-casein,        fibrinogen or thrombospondin. The amino acid sequences of        α₂-antiplasmin, fibronectin, beta-casein, fibrinogen and        thrombospondin can be found in the following references:        α₂-antiplasmin precursor [M. Tone et al., J. Biochem, 102, 1033,        (1987)]; beta-casein [L. Hansson et al, Gene, 139, 193, (1994)];        fibronectin [A. Gutman et al, FEBS Lett., 207, 145, (1996)];        thrombospondin-1 precursor [V. Dixit et al, Proc. Natl. Acad.        Sci., USA, 83, 5449, (1986)]; R. F. Doolittle, Ann. Rev.        Biochem., 53, 195, (1984);    -   peptides which are substrates or inhibitors of angiotensin, such        as:

angiotensin II (SEQ ID NO: 5) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe(E. C. Jorgensen et al, J. Med. Chem., 1979, Vol 22, 9, 1038-1044)[Sar, Ile] Angiotensin II: (SEQ ID NO: 6)Sar-Arg-Val-Tyr-Ile-His-Pro-Ile(R. K. Turker et al., Science, 1972, 177, 1203). Angiotensin I:(SEQ ID NO: 7) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu.

Preferably the peptides of the present invention comprise antiplasmin orangiotensin II peptides. Antiplasmin peptides comprise an amino acidsequence taken from the N-terminus of:

(i) α₂-antiplasmin, (SEQ ID NO: 8)i.e. NH₂-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu-Lys-OH

or variants of this in which one or more amino acids have beenexchanged, added or removed such as:

(SEQ ID NO: 9)NH₂-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu-Lys-Gly-OH,(SEQ ID NO: 10)NH₂-Asn-Gln-Glu-Ala-Val-Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu-Lys-Gly-OH,(SEQ ID NO: 11) NH₂-Asn-Gln-Glu-Gln-Val-Gly-OH; or (ii) casein(SEQ ID NO: 12) i.e. Ac-Leu-Gly-Pro-Gly-Gln-Ser-Lys-Val-Ile-Gly.

Synthetic peptides of the present invention may be obtained byconventional solid phase synthesis, as described in P. Lloyd-Williams,F. Albericio and E. Girald; Chemical Approaches to the Synthesis ofPeptides and Proteins, CRC Press, 1997.

Suitable enzyme substrates, antagonists or inhibitors include glucoseand glucose analogues such as fluorodeoxyglucose; fatty acids, orelastase, Angiotensin II or metalloproteinase inhibitors. A preferrednon-peptide Angiotensin II antagonist is Losartan.

Suitable synthetic receptor-binding compounds include estradiol,estrogen, progestin, progesterone and other steroid hormones; ligandsfor the dopamine D-1 or D-2 receptor, or dopamine transporter such astropanes; and ligands for the serotonin receptor.

The biological targeting moiety is preferably of molecular weight ofless than 5000, most preferably less than 4000, ideally less than 3000.Preferred biological targeting moieties are 3-20 mer peptides; or enzymesubstrates, antagonists or inhibitors.

The nature of the linker group (Z)_(q) can also be used to modify thebiodistribution of the metal complex when the complex is used as aradiopharmaceutical. Thus, e.g. the introduction of ether groups in(Z)_(q) will help to minimise plasma protein binding. When (Z)_(q)comprises a polyethyleneglycol (PEG) building block or a peptide chainof 1 to 10 amino acid residues, the linker group may function to modifythe pharmacokinetics and blood clearance rates of the imaging agent invivo. Such “biomodifier” linker groups may accelerate the clearance ofthe radiometal imaging agent from background tissue, such as muscle orliver, and/or from the blood, thus giving a better diagnostic image dueto less background interference. A biomodifier linker group may also beused to favour a particular route of excretion, e.g. via the kidneys asopposed to via the liver.

When (Z)_(q) comprises a peptide chain of amino acid residues, the aminoacid residues are preferably chosen from glycine, lysine, aspartic acid,glutamic acid or serine. The number of amino acids in the peptide chainis preferably 1 to 10, most preferably 1 to 3.

When (Z)_(q) comprises a PEG moiety, it preferably comprises a group offormula (—OCH₂CH₂O—)_(w) where w is an integer of value 3 to 25. Theinteger w is preferably 6 to 22. An especially preferred PEG-containing(Z)_(q) group is a unit derived from polymerisation of the monodispersePEG-like structure, 17-amino-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoicacid Formula Ia:

wherein p is an integer from 1 to 10.

Each E group is preferably an optionally substituted C₃₋₆nitrogen-containing heteroaryl ring. Preferred E groups are selectedfrom pyridyl, imidazoyl and pyrazoyl, with 2-pyridyl being especiallypreferred.

A preferred embodiment of Formula I is where each of R¹, R² and R³ are Egroups, with the preferred E groups as described above. In thisembodiment R¹, R² and R³ are preferably each the same E group, whichgives a symmetric tripodal ligand, and are most preferably each2-pyridyl.

R⁴ is preferably a functionalised acyl substituent of formula—NH(CO)—(Z)_(x)—Y, where x is an integer of value 0 to 9. Such acylsubstituents are preferred since they permit facile functionalisation ofthe ligand at the R⁴ position, and the acyl group does not compete withthe donor atoms of the R¹, R² and R³ groups. Such competition can be aproblem when R⁴ is amino (i.e. q=0 and Y is amino), since the aminegroup can also form a favoured 5-ring chelate upon metal coordination.

In a preferred metal complex of the ligand of Formula I, the metalco-ordinates via the A atom of the R¹ group and the nitrogen atom of theheteroaryl rings of the E groups, such that the ligand acts as atripodal chelating agent.

The preferred embodiments of the first aspect of the invention outlinedabove are metal complexes with an oxidation state of +1. These complexesare of particular interest when the metal is technetium because of thelow kinetic inertness of the d⁶-low spin Tc(I) ion. Furthermore, it ispossible to derivatise the ligand with a range of biological targetingmolecules allowing use of the resulting complexes in a range ofapplications. A key advantage of the synthetic protocol is that thetripodal ligands can be readily modified to produce both neutral andcharged complexes. Thus, in Formula (I), when R¹ is —CH₂(AR⁵), A ispreferably S. When R¹ is —CH₂(SR⁵), and an uncharged ligand is desired,R⁵ is preferably C₁₋₃ alkyl and is most preferably —CH₂(SCH₃),—CH₂(SC₂H₅) or —CH₂(SC₃H₇). When R¹ is —CH₂(SR⁵), and a negativelycharged ligand is desired, R⁵ is preferably H, i.e. R¹ is —CH₂SH.

The radiometal complexes of the present invention may be prepared byreaction of a solution of the radiometal in the appropriate oxidationstate with the ligand of Formula I at the appropriate pH. The solutionmay preferably contain a ligand which complexes weakly to the metal(such as gluconate or citrate) i.e. the radiometal complex is preparedby ligand exchange or transchelation. Such conditions are useful tosuppress undesirable side reactions such as hydrolysis of the metal ion.When the radiometal ion is ^(99m)Tc, the usual starting material issodium pertechnetate from a ⁹⁹Mo generator. Technetium is present in^(99m)Tc-pertechnetate in the Tc(VII) oxidation state, which isrelatively unreactive. The preparation of technetium complexes of loweroxidation state Tc(I) to Tc(V) therefore usually requires the additionof a suitable pharmaceutically acceptable reducing agent such as sodiumdithionite, sodium bisulphite, ascorbic acid, formamidine sulphinicacid, stannous ion, Fe(II) or Cu(I), to facilitate complexation. Thepharmaceutically acceptable reducing agent is preferably a stannoussalt, most preferably stannous chloride, stannous fluoride or stannoustartrate.

Further details on the preparation of ^(99m)Tc metal complexes are givenby Mease et al [Semin. Nucl. Med., 31(4), 278-285 (2001)]. Furtherdetails on the preparation of radioactive rhenium metal complexes aregiven by Hashimoto et al [Chapter 9 pages 275-291 in “Technetium andRhenium: Their Chemistry and its Applications”, Yoshihara and Omori(Eds), Springer, Berlin (1996)]. A preferred method of preparation of^(99m)Tc complexes of the present invention is via the use oflyophilised kits, as described in the fifth embodiment below.

In a second aspect, the present invention provides a metal complex ofFormula II:

[LMX₃]^(n)  (II)

where L is a ligand of Formula I as defined for the first embodiment,

M is a radioactive isotope of technetium or rhenium;

X is selected from CO, halogen, phosphine or isonitrile, and

n is the charge on the metal complex, and can be −1, 0, +1 or +2.

When X is an isonitrile, it is suitably a monodentate isonitrile. Whenall three X groups are phosphines, the three X donor atoms may be madeup of three monodentate phosphines, a single tridentate phosphine or amixture of a monodentate and a bidentate phosphine.

The charge on the metal complex (n) of Formula II will depend on thedonor atoms present in the ligand (L) and the identity of X. Thus, e.g.when X is CO, phosphine or isonitrile these are neutral donors and donot affect the charge. When X is halogen, however, these are negativelycharged donors. When R¹ of the ligand of Formula I is —CH₂SH it isanticipated that metal complexation occurs via a thiolate anion, i.e.the thiol is deprotonated so that the ligand carries a single negativecharge.

Metal complexes of Formula II can be prepared as described for the firstembodiment (above), plus the methods of Barrera et al [Inorg. Chem., 35,335-341 (1996)].

A preferred X group of Formula II is CO (i.e. a carbonyl group). Thiscorresponds to the charged complex of Formula Ha and the neutral complexof Formula IIb as described below:

[LM(CO)₃]^(n)  (IIa)

where: M is as defined in Formula II;

L is a ligand of Formula I as defined above, wherein:

R¹ is an E group or is —CH₂(AR⁵) wherein R⁵ is C₁₋₆ alkyl;

R⁴ is amino or —(Z)_(q)—Y wherein Z, q and Y are as defined in claim 1.

n is +1 or +2.

Preferred metal complexes of Formula IIa comprise the preferred ligandsas described for the first embodiment (above). Preferably n is +1.Preferably, R¹, R² and R³ in Formula IIa are each 2-pyridyl, and R⁴ is afunctionalised acyl substituent of formula —NH(CO)—(Z)_(x)—Y, where x isan integer of value 0 to 9 as described above for Formula II. When Y isnot a biological targeting moiety, Y is preferably amino, phenyl,pyridyl, piperidinyl, or imidazoyl. Most preferably, R⁴ is —NH(CO)Ph,—NH(CO)(CH₂)₃—(CO)NHCH₂Ph,

—NH(CO)(3-pyridyl), —NH(CO)(4-pyridyl), —NH(CO)CH₂-piperidinyl,—NH(CO)NH₂, or

—NH(CO)(2,4-imidazoyl).

A further preferred metal complex of Formula II is a neutral complex ofFormula IIb:

LM(CO)₃  (IIb)

wherein:

M is as defined in Formula II;

L is a ligand of Formula I wherein R¹ is —CH₂SH or —CH₂OH;

R² and R³ and R⁴ are as defined for Formula I.

Preferably, R¹ in Formula IIb is —CH₂SH and R² and R³ of L are each2-pyridyl.

The metal complex is neutral as a result of deprotonation of —CH₂SH or—CH₂OH upon coordination to the metal.

In Formulae II, IIa and IIb M is preferably ^(94m)Tc, ^(99m)Tc, ¹⁸⁶Re or¹⁸⁶Re and is most preferably ^(99m)Tc.

Where each X is CO and M is ^(99m)Tc, the complexes of the second aspectof the invention can be conveniently prepared from the^(99m)Tc(CO)₃((H₂O)₃ ⁺ precursor as described by Schibli et al [Bioconj.Chem., 11, p 345-351 (2000)] and Kothari et al [Appl. Rad. Isotop., 58,543-549 (2003)]. The three water molecules of the precursor can beeasily replaced by donor atoms of the ligand of Formula I in order toform Tc complexes of the invention. The ^(99m)Tc(CO₃)(H₂O)₃ ⁺ precursorfor non-clinical use may also be obtained using an Isolink™ kit(Mallinckrodt, Petten, Netherlands). Subsequent reaction of[^(99m)Tc(CO₃)(H₂O)₃]⁺ with a ligand of the invention results in theformation of a ^(99m)Tc complex of the invention. The correspondingrhenium metal complexes can be prepared by the methods of Pietzsch et al[Bioconj. Chem., 11, p 414-424 (2000)].

A number of examples of metal complexes of Formula IIa are illustratedin FIG. 1.

In a third aspect, the present invention provides a ligand as defined inFormula I, with the proviso that when R¹ is —CH₂SCH₃ or comprises a2-pyridyl or 2-quinoline ring, and R² and R³ each comprise a 2-pyridylring, then R⁴ is not H, —CH₂OH, C₁₋₄ alkyl, —NH₂ or

—NH(CO)R^(x), where R^(x) is C₁₋₁₂ alkyl, C₁₋₄ carboxyalkyl or—C₆H₄SO₃H.

One preferred class of ligand of the third embodiment is those in whichR¹ is —CH₂SH or

—CH₂OH. More preferred such ligands are those wherein either (i) R¹ is—CH₂SH, and R² and R³ each comprise a 2-pyridyl group; or (ii) whereinR¹, R² and R³ each comprise a 2-pyridyl group. Most preferred ligandsare those wherein R¹, R² and R³ each comprise a 2-pyridyl group.

Preferred R⁴ groups of the third embodiment are those in which Y iseither —(CH₂)_(m)NH₂ where m is an integer of value 1 to 6, preferably 2to 4; or is a biological targeting moiety, i.e. conjugates of theligands with a biological targeting moiety. Additional preferred R⁴groups are those wherein Y is amino-substituted phenyl, pyridyl,piperidyl, or imidazoyl.

The ligands of the present invention can be prepared by the method ofArnold et al [JCS Dalton Trans., 736-746 (2001)]. When R¹, R² and R³each comprise a 2-pyridyl group and R⁴ is H, the bridgehead proton (R⁴group) can be deprotonated with butyl lithium, and subsequentlyalkylated with alkyl halides as is taught by Kodera et al [Inorg. Chem.,42, 1193-1203 (2003)]. Functionalised versions thereof, includingconjugates with biological targeting moieties can also be prepared viathe bifunctional chelate approach. Thus, it is well known to prepareligands or chelating agents which have attached thereto a functionalgroup (“bifunctional linkers” or “bifunctional chelates” respectively).Functional groups that have been attached include: amine, thiocyanate,maleimide and active esters such as N-hydroxysuccinimide orpentafluorophenol. Ligand 1 of the present invention is an example of anamine-functionalised bifunctional chelate. Bifunctional chelatessuitable for complexation to a technetium or rhenium tricarbonyl coreare described by Stichelberger et. al [Nucl. Med. Biol., 30, 465-470(2003)]. Such bifunctional chelates can be reacted with suitablefunctional groups on the biological targeting moieties to form thedesired conjugate. Suitable functional groups on the compounds to beconjugated include:

carboxyls (for amide bond formation with an amine-functionalisedbifunctional chelator);

amines (for amide bond formation with an carboxyl- or activeester-functionalised bifunctional chelator);

halogens, mesylates and tosylates (for N-alkylation of anamine-functionalised bifunctional chelator) and

thiols (for reaction with a maleimide-functionalised bifunctionalchelator).

Examples 1 to 4 provide further syntheses of ligands of the invention.

In a fourth aspect, the present invention provides a radiopharmaceuticalcomprising the metal complex of the invention in any of the aspects andembodiments described above together with a biocompatible carrier in aform suitable for mammalian, especially human, administration. The“biocompatible carrier” is a fluid, especially a liquid, in which theimaging agent can be suspended or dissolved, such that the compositionis physiologically tolerable, i.e. can be administered to the mammalianbody without toxicity or undue discomfort. The biocompatible carrier issuitably an injectable carrier liquid such as sterile, pyrogen-freewater for injection; an aqueous solution such as saline (which mayadvantageously be balanced so that the final product for injection iseither isotonic or not hypotonic); an aqueous solution of one or moretonicity-adjusting substances (e.g. salts of plasma cations withbiocompatible counterions), sugars (e.g. glucose or sucrose), sugaralcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or othernon-ionic polyol materials (e.g. polyethyleneglycols, propylene glycolsand the like).

Such radiopharmaceuticals are suitably supplied in either a containerwhich is provided with a seal which is suitable for single or multiplepuncturing with a hypodermic needle (e.g. a crimped-on septum sealclosure) whilst maintaining sterile integrity. Such containers maycontain single or multiple patient doses. Preferred multiple dosecontainers comprise a single bulk vial (e.g. of 10 to 30 cm³ volume)which contains multiple patient doses, whereby single patient doses canthus be withdrawn into clinical grade syringes at various time intervalsduring the viable lifetime of the preparation to suit the clinicalsituation. Pre-filled syringes are designed to contain a single humandose, and are therefore preferably a disposable or other syringesuitable for clinical use. The pre-filled syringe may optionally beprovided with a syringe shield to protect the operator from radioactivedose. Suitable such radiopharmaceutical syringe shields are known in theart and preferably comprise either lead or tungsten.

Preferred radiopharmaceuticals of the present invention comprise thetechnetium radioisotopes ^(99m)Tc or ^(94m)Tc, most preferably ^(99m)Tc.When the technetium isotope is ^(99m)Tc, a radioactivity contentsuitable for a diagnostic imaging radiopharmaceutical is in the range180 to 1500 MBq of ^(99m)Tc, depending on the site to be imaged in vivo,the uptake and the target to background ratio.

Where X of Formula II is CO, the ^(99m)Tc radiopharmaceuticals of thepresent invention may be prepared by first preparing[^(99m)Tc(CO₃)(H₂O)₃]⁺ as described above. Subsequent reaction of[^(99m)Tc(CO₃)(H₂O)₃]⁺ with a ligand of the invention results in theformation of a ^(99m)Tc complex of the invention. Both steps are carriedunder sterile conditions or terminal sterilisation is employed.

In a fifth aspect, the present invention provides a non-radioactive kitfor the preparation of the radiopharmaceutical composition describedabove, which comprises the ligand of Formula (I), as defined in thefirst embodiment. When the radiometal is ^(99m)Tc, the kit suitablyfurther comprises a biocompatible reductant. Such kits are designed togive sterile radiopharmaceutical products suitable for humanadministration, e.g. via direct injection into the bloodstream. For^(99m)Tc, the kit is preferably lyophilised and is designed to bereconstituted with sterile ^(99m)Tc-pertechnetate (TcO₄ ⁻) from a^(99m)Tc radioisotope generator to give a solution suitable for humanadministration without further manipulation. Suitable kits comprise acontainer (e.g. a septum-sealed vial) containing the ligand or chelatorconjugate in either free base or acid salt form, together with abiocompatible reductant such as sodium dithionite, sodium bisulphite,ascorbic acid, formamidine sulphinic acid, stannous ion, Fe(II) orCu(I). The biocompatible reductant is preferably a stannous salt such asstannous chloride or stannous tartrate. Alternatively, the kit mayoptionally contain a metal complex which, upon addition of theradiometal, undergoes transmetallation (i.e. metal exchange) giving thedesired product.

The non-radioactive kits may optionally further comprise additionalcomponents such as a transchelator, radioprotectant, antimicrobialpreservative, pH-adjusting agent or filler. The “transchelator” is acompound which reacts rapidly to form a weak complex with technetium,then is displaced by the ligand. This minimises the risk of formation ofreduced hydrolysed technetium (RHT) due to rapid reduction ofpertechnetate competing with technetium complexation. Suitable suchtranschelators are salts of a weak organic acid, i.e. an organic acidhaving a pKa in the range 3 to 7, with a biocompatible cation. Suitablesuch weak organic acids are acetic acid, citric acid, tartaric acid,gluconic acid, glucoheptonic acid, benzoic acid, phenols or phosphonicacids. Hence, suitable salts are acetates, citrates, tartrates,gluconates, glucoheptonates, benzoates, phenolates or phosphonates.Preferred such salts are tartrates, gluconates, glucoheptonates,benzoates, or phosphonates, most preferably phosphonates, mostespecially diphosphonates. A preferred such transchelator is a salt ofMDP, i.e. methylenediphosphonic acid, with a biocompatible cation.

By the term “radioprotectant” is meant a compound which inhibitsdegradation reactions, such as redox processes, by trappinghighly-reactive free radicals, such as oxygen-containing free radicalsarising from the radiolysis of water. The radioprotectants of thepresent invention are suitably chosen from: ascorbic acid,para-aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid (i.e.2,5-dihydroxybenzoic acid) and salts thereof with a biocompatible cationas described above.

By the term “antimicrobial preservative” is meant an agent whichinhibits the growth of potentially harmful micro-organisms such asbacteria, yeasts or moulds. The antimicrobial preservative may alsoexhibit some bactericidal properties, depending on the dose. The mainrole of the antimicrobial preservative(s) of the present invention is toinhibit the growth of any such micro-organism in the radiopharmaceuticalcomposition post-reconstitution, i.e. in the radioactive diagnosticproduct itself. The antimicrobial preservative may, however, alsooptionally be used to inhibit the growth of potentially harmfulmicro-organisms in one or more components of the non-radioactive kit ofthe present invention prior to reconstitution. Suitable antimicrobialpreservative(s) include: the parabens, i.e. methyl, ethyl, propyl orbutyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol;cetrimide and thiomersal. Preferred antimicrobial preservative(s) arethe parabens.

The term “pH-adjusting agent” means a compound or mixture of compoundsuseful to ensure that the pH of the reconstituted kit is withinacceptable limits (approximately pH 4.0 to 10.5) for human or mammalianadministration. Suitable such pH-adjusting agents includepharmaceutically acceptable buffers, such as tricine, phosphate or TRIS[i.e. tris(hydroxymethyl)aminomethane], and pharmaceutically acceptablebases such as sodium carbonate, sodium bicarbonate or mixtures thereof.When the conjugate is employed in acid salt form, the pH adjusting agentmay optionally be provided in a separate vial or container, so that theuser of the kit can adjust the pH as part of a multi-step procedure.

By the term “filler” is meant a pharmaceutically acceptable bulkingagent which may facilitate material handling during production andlyophilisation. Suitable fillers include inorganic salts such as sodiumchloride, and water soluble sugars or sugar alcohols such as sucrose,maltose, mannitol or trehalose.

In a sixth aspect, the present invention relates to use of theradiopharmaceutical of the invention, wherein the radiometal is a gammaemitter, for SPECT imaging. Following administration of theradiopharmaceutical to a subject, the distribution of theradiopharmaceutical within said subject is detected by means of a gammacamera, which produces an image indicative of said distribution.

The invention is illustrated by the non-limiting Examples detailedbelow. Examples 1 to 4 provide the syntheses of Ligands 2 to 8. Example5 provides a method of preparation of a ^(99m)Tc-tricarbonyl complex ofLigand 6. The radiochemical purity (RCP) was 98%. Example 4 provides themethod of preparation of the ^(99m)Tc-tricarbonyl complexes of Ligands1, 2, 4, 5 and 6.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows several metal complexes of the invention.

FIG. 2 summarises the chemical structures of the ligands of theExamples.

EXPERIMENTAL EXAMPLES

Ligand 1 [i.e. 1,1,1-tris(pyridin-2-yl)methylamine] was prepared by themethod of Arnold et al [JCS Dalton Trans., 736-746 (2001)].

Example 1 Synthesis of Ligand 2

Benzoic anhydride (280 mg, 1.24 mmol) was added in one portion to astirred solution of Ligand 1 (250 mg 0.95 mmol) in CH₂Cl₂ (2 ml) at roomtemperature. Stirring was continued for 24 hours and the reactionmixture diluted with CH₂Cl₂ (18 ml). This solution was washed withsaturated sodium bicarbonate (20 ml×2) and water (20 ml×2), and thendried (MgSO₄), filtered and the solvent evaporated to leave a yellowresidue. Recrystallisation of this material from a CH₂Cl₂/pentanemixture gave the product (180 mg, 52%) as a yellow powder, m p 233-234°C.

¹H NMR (CDCl₃): δ_(H) 7.10-7.17 (3H, m, 5-H), 7.41-7.51 (3H, m, 4-H),7.59-7.64 (6H, m, 3-H [×2], meta-H [×2], para-H), 8.0 (2H, dd, J=7 and 2Hz, ortho-H [×2]), 8.53 (3H, br d, J=5 Hz, 6-H [×3]).

Mass Spec (ES) m/e:—Calculated for C₂₃H₁₉N₄O (M+H), 367; Found 367.

Example 2 Synthesis of Ligands 3 and 4

Thionyl chloride (5.0 g, 42 mmol) and nicotinic acid (620 mg, 5.0 mmol)were heated under reflux for 3 hours and the excess reagent then removedunder reduced pressure to leave a white residue. This residue wasco-evaporated with toluene (25 ml×2) under reduced pressure to leave awhite powder. This resulting solid was added in 50 mg portions withstiffing to a solution of Ligand 1 (250 mg, 0.95 mmol) in CH₂Cl₂ (10 ml)containing triethylamine (1.10 g, 10.0 mmol). After stiffing at roomtemperature for 16 hours the solution was washed with water (20 ml×2),dried (MgSO₄), filtered and the solvent removed under reduced pressureto leave a dark brown residue. This residue was purified bychromatography on silica gel eluting with a CH₂Cl₂/MeOH mixture (20:1).The required product was isolated as a yellow solid (r_(f)=0.25) whichwas recrystallised from CH₂Cl₂/Pentane to give a pale yellow powder(Yield 210 mg, 60.0%), m p 206-207° C.

¹H NMR (CDCl₃): δ_(H) 7.14 (3H, dt, J=5.0 and 2.7 Hz, 5-H [×3], 7.35(1H, dd, J=8.0 and 5.0 Hz, 5′-H), 7.58-7.61 (3H, m 4-H [×3]), 7.62 (3H,dt, J=8.2 and 1.5 Hz, 3-H [×3]), 8.23 (1H, dt, J=8.0 and 2.0 Hz, 4′-H),8.51 (3H, br d, J=5 Hz, 6-H [×3]), 8.71 (1H, dd, J=5.0 and 1.5 Hz,6′-H), 9.25 (1H, d, J=2.0 Hz, 2′-H), 10.4 (1H, s, NH).

Mass Spec (ES) m/e: Calculated for C₂₂H₁₈N₅O (M+H), 368; Found 368.

Ligand 4 was prepared in an analogous manner from isonicotinic acid asthe starting material, giving a pale yellow powder (140 mg, 40.0%). m p183-185° C.

¹H NMR (CDCl₃): δ_(H) 7.15 (3H, dt, J=5.0 and 1.5 Hz, 5-H [×3],7.57-7.66 (6H, m 4-H [×3], 3-H [×3], 7.80 (2H, d, J=5.9 Hz, 2′-H, 6′-H)8.51 (3H, d, J=5 Hz, 6-H [×3], 8.74 (2H, d, J=5.9 Hz, 3′-H, 5′-H), 10.45(1H, bs, NH).

Mass Spec (ES) m/e:—Calculated for C₂₂H₁₈N₅O (M+H), 368; Found 368.

Example 3 Synthesis of Ligand 5

Step (a): Ethyl 2-(piperidin-1-yl)-acetate.

Ethyl bromoacetate (8.5 g, 50.9 mmol) was added dropwise to a stirredsolution of piperidine (4.3 g, 50.5 mmol) and triethylamine (5.5 g, 54.4mmol) in chloroform (20 ml) at such a rate as to maintain a gentlereflux. After the addition was complete stirring was continued for 2hours and the reaction mixture washed with water (20 ml×3). Thechloroform layer was dried (MgSO₄) filtered and the solvent evaporatedto leave a yellow oil. This oil was passed through a short column ofsilica eluting with a CH₂Cl₂/Pet Ether 40:60 mixture (1:1). Removal ofthe solvent from the resulting solution gave the required product as apale yellow oil (Yield 5.5 g, 64%).

¹H NMR (CDCl₃):—δ_(H) 1.20 (3H, t, J=7.0 Hz, CH₃), 1.40 (2H, m, 4′-H),1.55 (4H, m, 3′-H 5′-H), 2.42 (4H, m, 2′-H 6′-H), 3.10 (2H, s, NCH₂CO)4.10 (2H, q, J=7 Hz, OCH₂).

Step (b): Piperidin-1-yl acetic acid hydrochloride.

Ethyl 2-(piperidin-1-yl)-acetate (5.5 g, 32.2 mmol), water (12 ml) and cHCl (3 ml) were heated under reflux for 20 hours. Volatile componentswere evaporated under reduced pressure to leave an off white solid. Thissolid was co-evaporated with toluene (50 ml×3) and dried in vacuo togive an off white powder (Yield 4.8 g, 83%).

¹H NMR (D₂O);—δ_(H) 1.42-1.46 (1H, m, 4′-H), 1.73-1.86 (5H, m, 2′-H 3′-H4′-H), 2.97-3.05 (2H, m, 2′-H 6′-H), 3.52-3.57 (2H, m, 2′-H 6′-H), 4.02(2H, s, NCH₂CO).

Step (c):2-(Piperidin-1-yl)-N-[1,1,1-tris(pyridyl-2-yl)methyl]-acetamide

Lithium hydroxide (85 mg, 3.5 mmol) and piperidin-1-yl acetic acidhydrochloride (300 mg, 1.68 mmol) were dissolved in methanol (10 ml) togive a colourless solution. The methanol was then was removed underreduced pressure and the colourless solid co-evaporated with tolueneuntil a free flowing powder remained. To this lithium salt was added dryDMF (10 ml), Ligand 1 (250 mg, 0.95 mmol), Pybop (510 mg, 0.98 mmol) andthe mixture warmed until a clear solution was obtained. Heat was removedand stirring continued for 16 hours. Volatile components were removedunder reduced pressure to leave an orange residue which was dissolved inCH₂Cl₂ (20 ml) and washed with water (20 ml×3). The organic layer wasdried (MgSO₄), filtered and the solvent evaporated to leave an orangeoil. This residue was purified by chromatography on silica gel elutingwith a CH₃CN/MeOH mixture (200:5). The required product was isolated(r_(f)=0.2) as a pale yellow oil which solidifies on standing (100 mg27%). m p 123-125° C.

¹H NMR (CDCl₃):—δ 1.45-1.50 (2H, m, 4′-H)), 1.65-1.63 (4H, m, 3′-H5′-H), 2.54-2.57 (4H, m, 2′-H 6′-H), 3.01 (2H, s, NCH₂CO), 7.12 (3H,ddd, J=7.5, 5.0 and 1.0 Hz, 5-H, [×3]) 7.46-7.50 (3H, m, 3-H [×3]), 7.60(3H, dt, J=7.5 and 5.0 Hz, 4-H [×3]), 8.50-8.53 (3H, m, 6-H [×3]), 10.61(1H, s, NH).

Mass Spec (ES) m/e:—Calculated for C₂₃H₂₆N₅O (M+H), 388 Found 388

Example 4 Synthesis of Ligands 6, 7 and 8

1H-Imidazole-4-carboxylic acid (200 mg, 1.8 mmol) and lithium hydroxide(45 mg, 1.9 mmol) were dissolved in methanol (10 ml) to give a clearsolution. Volatile components were removed under reduced pressure andthe residue co-evaporated with toluene until a free flowing powder wasobtained. To the lithium salt was added dry DMF (10 ml), Ligand 1 (250mg, 0.95 mmol) and Pybop (510 mg, 0.98 mmol). The mixture was warmeduntil a clear orange solution was obtained. Heat was then removed andthe solution stirred for 16 hours. The solvent was removed by rotaryevaporation to leave a viscous orange residue which was purified bychromatography on silica gel eluting with CH₃CN/MeOH (95:5). The titlecompound (100 mg, 29%) was isolated (r_(f)=0.3) as a white powder mp>250° C.

¹H NMR (d⁶ DMSO):—δ_(H) 5.76 (1H, s, NH imid), 7.23-7.28 (3H, m, 5-H[×3], 7.44-7.47 (3H, m, 3-H [×3], 7.53 (1H, s, C—H, imid), 7.72 (3H, bt,J=7 Hz, 4-H [×3], 7.82 (1H, s, C—H imid), 8.50 (3H, m, 6-H [×3]), 10.46(1H, s, NH).

Mass Spec (ES) m/e:—Calculated for C₂₀H₁₇N₆O (M+H), 357; Found 357.

Ligand 7 was prepared in an analogous manner from 4-pyridylacetic acidhydrochloride giving a colourless powder (Yield 40 mg, 11%), m pt134-135° C.

¹H NMR (CDCl₃): δ_(H) 3.70 (2H, s, CH₂), 7.13 (3H, m, 5-H [×3], 7.26(2H, d, J=5.0 3-H, 5-H), 7.43 (3H, d, J=6 Hz 3-H [×3], 7.57 (3H, t, J=6Hz 4-H [×3], 8.47 (3H, d, J=5 Hz, 6-H [×3], 8.53 (2H, d, J=5.0 Hz, 2-H,6-H), 9.49 (1H, bs, NH).

Mass Spec (ES) m/e:—Calculated for C₂₃H₂₀N₅O (M+H), 382; Found 382

Ligand 8 was prepared in an analogous manner from 3-Pyridyl acetic acidhydrochloride giving a fawn coloured powder (180 mg, 54%), m p 165-166°C.

¹H NMR (CDCl₃): δ_(H) 3.68 (2H, s, CH₂) 7.08 (3H, ddd, J=7.5, 5.0 and1.0 Hz, 5-H [×3], 7.19 (1H dd, J=7.5 and 5.0 Hz 5′-H), 7.43 (3H, m, 3-H[×3]), 7.53 (3H, dt, J=7.5 and 5.0 4-H [×3], 7.64 (1H, bd, J=7.5 Hz4′-H), 8.43 (3H, m, 6-H [×3], 8.46 (1H, dd, J=7.5 and 1.5 Hz, 6′-H),8.56 (1H, d, J=1.5 Hz, 2′-H), 9.49 (1H, s, NH).

Mass Spec (ES) m/e:—Calculated for C₂₃H₂₀N₅O (M+H), 382; Found 382.

Example 5 Preparation of the ^(99m)Tc Tricarbonyl Complex of Ligand 6

1 mL of TcO₄ ⁻ eluted from a generator was added to a Tc-carbonyl kit(Isolink™, Mallinckrodt, Petten, Netherlands). Activity was measured at983 MBq. Approximately 4 mL headspace was removed from the vial, whichwas then heated to approx 100° C. for 30 minutes. The vial was allowedto cool, and the pressure was equalised using bleed needle. 50-100 ug ofLigand 6 was added before removal of 4 mL headspace. The vial was heatedto approx 100° C. for 30 minutes and then allowed to cool, beforeequalising the pressure of the vial using bleed needle. The pH wasmeasured to be approximately 11.5 and the radiochemical purity (RCP)determined by high performance liquid chromatography (HPLC) was 98%.

Two HPLC methods were used as follows:

Method A

Solvent A=0.1% TFA in water, solvent B=0.1% TFA in acetonitrile

Method B

Solvent A=0.2% 880 ammonia solution, solvent B=acetonitrile

For both methods, the following gradient was used:

Time (min) % B 0 20 5 45 14 45 15 100 19 100 21 20 23 20

Example 6 Preparation of ^(99m)Tc Tricarbonyl Complexes of Ligands 1, 2,4, 5 and 6

1 mL TcO₄ ⁻ eluted from a generator was added to a Tc-carbonyl kit(Isolink™, Mallinckrodt, Petten, Netherlands). The activity was measuredas 2.88 Gq. Approximately 4 mL headspace was removed from the vial,which was then heated to approximately 100° C. for 30 minutes. The vialwas allowed to cool, and the pressure equalised using bleed needle.

50-100 ug (in 0.1 mL MeOH) of Ligand was added to a nitrogen filledvial, followed by 0.7 mL 0.4M phosphate buffer (pH approx 7.5). 0.2 mLTc(CO)₃(H₂O)₃]⁺ prepared from the kit was then added and the activitywas measured as 593 Mq. The vial was heated to approx 100° C. for 30minutes before being allowed to cool, and the pressure equalised using ableed needle. The pH was approximately 8.5 and the RCP for each complexas determined by HPLC (described in Example 5) was as follows:

^(90 m)Tc Complex RCP (%) Ligand 1 94 Ligand 2 65 Ligand 4 40 Ligand 587 Ligand 6 90

1. A metal complex of a ligand of Formula I:

wherein: the metal is a radioactive isotope of either technetium orrhenium; R¹-R³ are independently selected from pyridyl, imidazoyl andpyrazoyl; and, R⁴ is —NH(CO)Ph, —NH(CO)(CH₂)₃—(CO)NHCH₂Ph,—NH(CO)(3-pyridyl), —NH(CO)(4-pyridyl), —NH(CO)CH₂-piperidinyl,—NH(CO)NH₂, or —NH(CO)(2,4-imidazoyl); and wherein not more than threeout of a possible five atoms of the ligand are involved in complexation.2-4. (canceled)
 5. The metal complex of claim 1 wherein R¹, R² and R³are each pyridyl.
 6. The metal complex of claim 1 wherein R¹, R² and R³are each 2-pyridyl.
 7. The metal complex of claim 1 wherein the metal isa radioactive isotope of technetium.
 8. The metal complex of claim 7wherein the radioactive isotope of technetium is ^(99m)Tc.
 9. A metalcomplex of Formula II:[LMX₃]^(n)  (II) wherein: L is a ligand of Formula I as defined in claim1; M is the radioactive isotope of technetium or rhenium; X is selectedfrom CO, halogen, phosphine, or isonitrile; and, n is the charge on themetal complex, and is −1, 0, +1 or +2.
 10. The metal complex of claim 9which is of Formula IIa:[LM(CO)₃]^(n)  (IIa) wherein L is a ligand of Formula I as defined inclaim 1, wherein: R¹, R² and R³ are each 2-pyridyl, and n is +1 or +2.11-15. (canceled)
 16. The metal complex of claim 9 wherein M is aradioactive isotope of technetium.
 17. The metal complex of claim 16wherein the radioactive isotope of technetium is ^(99m)Tc.
 18. A ligandof Formula I as defined in claim
 1. 19-21. (canceled)
 22. The ligand ofclaim 18 wherein R¹, R² and R³ each comprise a 2-pyridyl group.
 23. Aradiopharmaceutical comprising the metal complex of claim
 1. 24-26.(canceled)
 27. A kit for the preparation of the radiopharmaceutical ofclaim 23 comprising a ligand of Formula I as defined in claim
 18. 28-29.(canceled)